Over the past 30 years, immunochemical assays have become increasingly widespread. The most sensitive type of immunoassay is the solid phase (heterogeneous) immunoassay. However, it is desirable to increase sensitivity even further in order to be able to detect analytes which are present in lower levels than is presently possible to detect and to be able to detect more accurately the concentrations of the analytes. Sensitivity of the solid phase immunoassay is limited by several factors, including non-specific binding (NSB) of the tracer to the solid phase. The NSB is a result of either direct adsorption of the tracer to the solid phase surface, or indirect adsorption, via binding to non-specific components of the assay mixture that are adsorbed to the surface of the solid phase. These non-specific components are at least partially different than the material to be assayed, but have affinity toward the tracer. The adsorption of the non-specific components can lead to decreased sensitivity and also to reduced accuracy. One way of reducing NSB is to use a process called immune complex transfer (ICT), which was pioneered by Ishikawa. (See Ishikawa et al, 185 Clinica Chimica Acta (1989) 223; and Kohno et al, 22 Clin Biochem (1989) 277.)
In a conventional heterogeneous immunoassay, the assayed material (analyte) is incubated with a labeled specific binder (tracer) and an unlabeled, solid phase-immobilized specific binder. The incubation is followed by washing the solid phase to remove unreacted tracer, and measuring the signal associated with the ternary complex: solid phase-immobilized specific binder/analyte/labeled specific binder. This measurement is done in the presence of the solid phase, and therefore includes the NSB. On the other hand, in the ICT assay, the ternary complex is eluted off of the solid phase and is measured in the absence of the solid phase and its associated NSB. Since some of the NSB is eluted as well, it is preferable to include in the ICT process a step in which the labeled complex is selectively recaptured with a new solid phase. This results in increased signal to noise ratio and higher assay sensitivity.
The ICT process requires elution of the immune complex under mild conditions, i.e. without breaking the bond formed between the analyte and the specific labeled binder. Also, the elution medium and elution conditions should not interfere with the recapture step. Ishikawa et al overcame this difficulty by constructing an immunochemical, reversible bridge between the solid phase specific binder and the solid phase, rather than employing the usual permanent attachment. They permanently immobilized to the solid phase an antibody against dinitrophenyl residues (anti-DNP), separately attached to the specific binder dinitrophenyl residues and then combined the two reagents to form a solid phase-immobilized specific binder. This provided a reversible immunochemical bond that could be broken by addition of excess dinitrophenyl-lysine (DNP-lys)
Although the use of solid phase immobilized anti-DNP/dinitrophenylated specific binder as the solid phase reagent by Ishikawa et al successfully reduced NSB, the method has two drawbacks: the major difficulty is the lengthy incubation with DNP-lys required for the elution step. Reducing the incubation time of this step is highly desirable in order to both obtain a quicker assay result and to minimized loss of sensitivity due to the time-dependent dissociation of the bond between the analyte and the labeled specific binder. A second difficulty is the added complexity, expense and instability associated with constructing the immunochemical bridge.